Kinetics of oxygen binding and subunit assembly for the hemoglobin alpha subunit

Abstract

A thorough kinetic characterization of the O 2-binding and self-association reactions of α-subunits of human hemoglobin A has been performed. All of the rate constants for a five step reaction model linking the monomer-dimer reaction to the O 2-binding steps have been determined for the first time. Our analysis of the ligand binding reaction shows that both monomer and dimer have nearly identical intrinsic O 2-association and dissociation rate constants and therefore identical affinities for oxygen. During this investigation we discovered a small absorbance difference between the oxy-monomer and oxy-dimer alpha-subunits. This difference spectrum enabled direct measurements of the αO 2 self-association reaction. We find an association rate constant of, 2.0 10 5 M −1s −1, similar to that for other subunit assembly processes in the hemoglobin system. Our results also suggest that the deoxy-subunit assembly kinetics must be similar to that for the oxy-subunit. These kinetic results together with the equilibrium constants obtained for these solution conditions by Ackers and coworkers provides, for the first time, a complete kinetic and thermodynamic description of all the intrinsic ligand binding and association reactions for alpha-subunits.